Sample Size

R02548045 蘇宸瑩D02548002 蔡靜雯

2014.12.03

Biocompatibility (ISO 10993)

Part 3. Tests for genotoxicity, carcinogenicity and reproductive toxicity

Part 4. Selection of tests for interactions with blood

Part 5. Tests for in vitro cytotoxicity

Part 10. Tests for irritation and delayed-type hypersensitivity

Part 11. Tests for systemic toxicity

Cytotoxicity (ISO 10993-5)

Sample preparationa) an extract of the test sampleb) the test sample itself.

Experiment I (N=1)

PU gel

(Cross-link: A > B)

Extraction: 37°C, 24 hours

Cell: human fibroblasts

(Seeding density: 2000/cm2)

n = 4

** p < 0.01 (Student’s t-test)Control A B

Abs

orba

nce

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7 **

Experiment I & II (N=2)

Problem:

How do we analyze these data for significant results?

Control A B

Abs

orba

nce

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

Control A BA

bsor

banc

e

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35** **

PCR used to amplify a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence.

Polymerase Chain Reaction (PCR)

Denaturation

Annealing Elongation

Reverse transcription PCR

DNA→ PCR (amplify) → Gel electrophoresis

Real-time PCR

Also called quantitative PCR, Q-PCR

Fluoroance

SYBR Green 1 dye

TagMan® probe

Amplification plot

Relative Quantification

Data analysis

Δ Ct = Ct(T) – Ct(E)

ΔΔCt = ΔCt(S) – ΔCt(Ctrl)

Copy number = 2 - ΔΔCt

Example:

T : Target geneE : Endogenous gene

(internal control)S : SampleCtrl : Control

c- Myc GAPDH Δ Ct Δ Δ Ct

T = 0 25 10 15 0

T = 12 hr 24 10 14 -1

T = 24 hr 23 11 12 -3

T = 48 hr 28 10 18 3

Experiment I & II (N=2)

Problem:

How do we analyze these data for significant results?

Control A B

Abs

orba

nce

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

Control A BA

bsor

banc

e

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35** **

　 control A B

　 OD cells OD /cells OD cells OD /cells OD cells OD /cells

　 0.601 95000 6.328E-06 0.583 94500 6.165E-06 0.442 90000 4.916E-06

　 0.584 94800 6.158E-06 0.594 94000 6.317E-06 0.461 88000 5.240E-06

　 0.599 95300 6.282E-06 0.594 95200 6.238E-06 0.479 89000 5.387E-06

　 0.577 94900 6.079E-06 0.581 95430 6.085E-06 0.447 86000 5.200E-06

Average 　 　 6.212E-06 　 　 6.201E-06 　 　 5.185E-06

exp./control 　 　 1　 　 　 0.998 　 　 0.835

　 control A B

　 OD cells OD /cells OD cells OD /cells OD cells OD /cells

　 0.299 72000 4.151E-06 0.313 70000 4.466E-06 0.265 75000 3.532E-06

　 0.296 72500 4.087E-06 0.318 68000 4.674E-06 0.258 76000 3.399E-06

　 0.318 71000 4.477E-06 0.282 69000 4.084E-06 0.278 78000 3.565E-06

　 0.301 72600 4.152E-06 0.305 73000 4.181E-06 0.272 75000 3.631E-06

Average 　 　 4.217E-06 　 　 4.351E-06 　 　 3.532E-06

exp./control 　 　 1　 　 　 1.032 　 　 0.838

Experiment I

Experiment II

Control A B

Rel

ativ

e O

.D./c

ells

(ex

p./c

ontr

ol)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

Control A B

Rel

ativ

e O

.D./c

ells

(ex

p./c

ontr

ol)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

Control A B

Rel

ativ

e O

.D./c

ells

(ex

p./c

ontr

ol)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

Experiment I

Experiment II

Experiment I + II